There is a significant polarity in the concentrations and redox potentials between the epithelial lining fluid (ELF) and plasma for reduced and oxidized glutathione (GSH/GSSG) and cysteine and cystine (Cys/CySS). Further, these thiol pairs are not in equilibrium in either compartment. Using methods that we developed, we demonstrated that the thiol/disulfide redox gradients between the mitochondria, nuclei, cytoplasm and secretory pathways are not in equilibrium and perturbation within these compartments regulates the function of redox-sensitive proteins. The remarkable differences in steady-state redox states among lung compartments and the changes in these redox states associated with risk of lung injury led us to hypothesize that disruption of the extracellular redox circuitry between the alveolar space and plasma will 1) alter organelle redox states, 2) activate redox-sensitive signaling, and 3) alter the susceptibility to lung injury. Equally important, we hypothesize that restoration of extracellular redox states will normalize organelle redox states and decrease the risk of injury. The focus of this application is to demonstrate that perturbations of the redox circuitry on the apical or basolateral surface do not simply reflect oxidant stress. Rather, these extracellular changes impact the redox state of the different organelles and initiate cell signaling independent of the reactive oxygen species generated. Using alveolar type I (AT1) cell and type II (AT2) cell lines and primary AT2 cell monolayers, we will determine if oxidation of the GSH/GSSG or Cys/CySS redox gradients on the apical alveolar surface will disrupt 1) the redox gradients on the basolateral surface, 2) the redox gradients in the mitochondria, cytoplasm, nuclei and the secretory pathway of AT1 and AT2 cells, and 3) redox signaling (Aim 1). In Aim 2, we will examine if disruption of organelle redox states induced by perturbations of redox circuitry on the apical alveolar surface will increase the risk of cytotoxin-induced injury. In Aim 3, we will determine if perturbations on the basolateral surface will disrupt 1) the redox gradient on the apical surface, 2) the redox gradients in the mitochondria, cytoplasm, nuclei and the secretory pathway of AT1 and AT2 cells, and 3) redox signaling. In Aim 4 we will examine the extent by which perturbations on the basolateral surface will increase the risk of injury. Finally, we will determine in Aims 3 and 4 how restoration of these gradients on the apical or basolateral surface will restore redox signaling and decrease the sensitivity to injury. These studies will provide unprecedented insight into how oxidative stress and changes in the GSH/GSSG or Cys/CySS ratio in the alveolar space is transmitted to organelles to cause oxidative stress within these different subcellular compartments. PUBLIC HEALTH RELEVANCE: There is a significant polarity in the thiol/disulfide concentrations and redox states between the alveolar lining fluid and plasma. This study will determine if disruption of this polarity between these two extracellular pools will alter the redox state of subcellular compartments of epithelial cells, the redox state of redox-sensitive proteins or the susceptibility to lung injury. The results of these proposed studies will provide an unprecedented knowledge concerning the subcellular compartmentalization of oxidative stress in the lung.